PEG-lipid conjugates for liposomes and drug delivery

ABSTRACT

New diacylglycerol-polyethylene glycol (DAG-PEG) conjugates are described. A variety of linkers between the PEG chain and glycerol backbone of the DAG-PEGs may be selected to optimize liposomal formulations of pharmaceuticals and cosmetics.

RELATED APPLICATIONS

This application claims priority to provisional U.S. patent application61/131,674 entitled “PEG-LIPID CONJUGATES FOR LIPOSOMES AND DRUGDELIVERY” and filed on Jun. 11, 2008; and to provisional United Statespatent application 61/135,515 entitled “PEG-LIPID CONJUGATES FORLIPOSOMES AND DRUG DELIVERY” and filed on Jul. 21, 2008.

FIELD OF THE INVENTION

This invention relates to lipids and liposomes. More particularly, thepresent invention relates to new PEG-lipid conjugates and their use tomake liposomes for drug delivery, cosmetics and other purposes.

BACKGROUND OF INVENTION

Polyethylenglycol (PEG) is widely used as a water soluble carrier forpolymer-drug conjugates. PEG is undoubtedly the most studied and appliedsynthetic polymer in the biomedical field [Duncan, R. Nature Rev. DrugDiscov. 2003, 2, 347-360]. As an uncharged, water-soluble, nontoxic,nonimmunogenic polymer, PEG is an ideal material for biomedicalapplications. Covalent attachment of PEG to biologically activecompounds is often useful as a technique for alteration and control ofbiodistribution and pharmacokinetics, minimizing toxicity of thesecompounds [Duncan, R. and Kopecek, J., Adv. Polym. Sci. 57 (1984),53-101]. PEG possesses several beneficial properties: very low toxicity[Pang, S. N. J., J. Am. Coil. Toxicol, 12 (1993), 429-456], excellentsolubility in aqueous solutions [Powell, G. M., Handbook of WaterSoluble Gums and Resins, R. L. Davidson (Ed.), Ch. 18 (1980),MGraw-Hill, New York], and extremely low immunogenicity and antigenicity[Dreborg, S, Crit. Rev. Ther. Drug Carrier Syst., 6 (1990), 315-365].The polymer is known to be non-biodegradable, yet it is readilyexcretable after administration into living organisms. In vitro studyshowed that its presence in aqueous solutions has shown no deleteriouseffect on protein conformation or activities of enzymes. PEG alsoexhibits excellent pharmacokinetic and biodistribution behavior.[Yamaoka, T., Tabata, Y. and Ikada, Y., J. Pharm. Sci. 83 (1994),601-606].

In the early developmental stage of PEGylation, the attention has beenfocused on the amino groups, which are the most represented groups inproteins and are the most suitable conjugation sites. Amino groups aregenerally exposed in an aqueous environment or other solvent, and can bemodified with a wide selection of chemical strategies. Severalconjugation strategies are now available, such as alkylation, whichmaintains the positive charge of the starting amino group because asecondary amine is formed, or acylation, accompanied by loss of charge.[Graham, L. M., Adv. Drug Deliv. Rev. 55 (2003), 1293-1302; Levy, Y. etal., J. Pediatr., (1988) 113, 312-317; Bailon, P. et al., Bioconjug.Chem., 12 (2001), 195-202; Wang, Y. S. et al., Adv. Drug Deliv. Rev. 54(2002), 547-570; Kinstler, O. B. et al., Pharm. Res., 13 (1996),996-1002; Wong, S. S., Chemistry of protein conjugation andcross-linking, p. 13 (1991), CRC Press; Caliceti, P. et al., J. Bioact.Comp. Polym. 8 (1993), 41-50]

Esters with PEG have been utilized in chemical modifications of drugs.PEG esters which have an electron withdrawing substituent (alkoxy) inthe a-position have proved to be especially effective linking groups inthe design of prodrugs since the substituent aids in the rapidhydrolysis of the ester carbonyl bond, thus releasing alcohols in acontinuous and effective manner. For instance, highly water solublePEG-5000 esters of paclitaxel were synthesized and shown to function asprodrugs, i.e., breakdown occurred in a predictable fashion in vitro.[R. B. Greenwald, A. Pendri, D. Bolikal, C. W. Gilbert, Bioorg. Med.Chem. Lett., 4 (1994), 2465-2470]. Studies also showed that amino acidconjugates appeared to be the most useful, reducing toxicity whileincreasing efficacy for most of the anticancer drugs [A. Pendri, C. D.Conover, R. B. Greenwald, Anti-Cancer Drug Design, 13 (1998), 387-395;R. B. Greenwald, A. Pendri, C. D. Conover, C. Lee, Y. H., Choe, C.Gilbert, A. Martinez, J. Xia, D. Wu, M. Hsue, Bioorg. Med. Chem., 6(1998), 551-562]

Thiol modification is another potentially useful strategy of PEGylation.For instance, nonessential amino acids in a protein sequence can bereplaced by cysteine residues and can be replaced almost anywhere. Suchmutant proteins have been generated to PEGylate therapeuticallyimportant drugs such as Granulocyte colony-stimulating factor (G-CSF) orhuman growth hormone (HGH) [Cox, G., Bolder Biotechnology, WO9903887;Berna, M. et al., 32nd Annual meeting & exposition of the controlledrelease society, 18-22 Jun. (2005), Abstract No. 415, Miami, USA]

Less specific linking strategies relied on the reduction of proteindisulphide bridges with the aim of exposing new thiol groups have beenused for the PEGylation of antibodies, as amino groups are not suitableas the modification sites because of the marked loss of recognition thatoccurs during the PEGylation procedure. However, disulphide bridges thatlink the IgG heavy chains can be cleaved, yielding an active Fab moietywith new exposed thiol groups where the sites of conjugation are alsolocalized far from the antibodies recognition site. [A. P. Chapman etal., Nat. Biotechnol., 17 (1999), pp. 780-783]

In recent years lipid conjugates to PEG have generated great interest,as a result of the discovery that incorporation of PEG-lipids intoliposomes yields preparations with superior performance in comparison toconventional liposomes. Such liposomes remain in the blood circulationfor extended periods of time and distribute through an organismrelatively evenly with most of the dose remaining in the bloodcompartment and only 10-15% of the dose in liver. This constitutes asignificant improvement over conventional liposomes. [Woodle, M. C. andLasic, D. D., Biochim. Biophys. Acta, 1113 (1992), 171-199]. In thesestudies, amide-linked mPEG-DSPE(1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine) was prepared bycoupling mPEG to the amino group of phosphatidyl ethanolamines [Parr, M.J., Ansell, S. M., Choi, L. S. and Cullis, P. R., Biochim. Biophys.Acta, 1195 (1994), 21-30] and the modified surface amino groups ofDSPC-DPPE-cholesterol vesicles were reacted with mPEG-tresylate afterliposome formation, instead of using a PEG-lipid conjugate to formliposomes [Senior, J., Delgado, C., Fisher, D., Tilcock, C. andGregoriadis, Biochim. Biophys. Acta, 1062 (1991), 77-82]. The attractivefeature of this approach is in its selective grafting of the polymer onthe exterior of the vesicles, thus avoiding the presence of mPEGresidues inside the liposomes. A similar study was reported, wheregrafting mPEG residues onto preformed liposomes, maleimido-PE-containingvesicles were prepared and then reacted with a thiol derivative of PEG[Herron, J. N., Gentry, C A., Davies, S. S., Wei. A. and Lin, J. N., J.Controlled Rel., 28 (1994), 155-166].

Despite all this progress, significant potential for improved liposomaldrug delivery remains. So far, few liposomal drugs have been approvedfor clinical use. Difficulties in obtaining suitable formulations ofmany drugs remain the challenges. Furthermore, it is desirable todevelop new methods and materials to improve manufacturing, celltargeting, and drug release.

BRIEF SUMMARY OF THE INVENTION

New diacylglycerol-polyethylene glycol (DAG-PEG) conjugates aredescribed. A variety of linkers between the PEG chain and glycerolbackbone of the DAG-PEGs may be selected to optimize liposomalformulations of pharmaceuticals and cosmetics.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows stability profiles in a low pH medium of PEG(n=12)-3-acetyl-1,2-rac-dioleoylglycerol, PEG(n=12)-3-acetamido-dioleoylglycerol and PEG(n=12)-3-N-(mercaptomethyl)-Propionamido-1,2-rac dioleoylglycerol.

FIG. 2 shows mouse IV PK (pharmacokinetic) profiles of DAG-PEGformulations of PEG-12-acetyl-GDO (glycerol dioleate) andPEG-2-acetamido-GDO of rifampicin, and a rifampicin solution containing5% dimethyl sulfoxide and 10% Cremophor EL.

FIG. 3 shows mouse oral PK profiles of DAG-PEG formulations ofPEG-12-acetyl-GDO and PEG-12-acetamido-GDO of rifampicin, a rifampicinsolution containing 5% dimethyl sulfoxide and 10% Cremophor EL.

FIG. 4 shows rifampicin levels in lung in a mouse model after the drugwas administered intravenously.

FIG. 5 shows rifampicin levels in liver in a mouse model after the drugwas administered intravenously.

FIG. 6 shows mouse IV PK profiles of posaconazole with formulation ofPEG-12-acetamido (N₃)-GDO (1:3 drug to lipid ratio) and PEG-12-acetamido(N₃)-GDM (1:5 drug to lipid ratio), POPC(1-Palmitoyl,2-oleoyl-sn-Glycero-3-phosphocholine, 1:1 drug to lipidratio) and a posaconale solution containing 5% dimethyl sulfoxide and10% Cremophor EL.

FIG. 7 shows mouse oral PK profiles of posaconazole with formulation ofPEG-12-acetamido (N₃)-GDO (1:3, drug to lipid ratio) andPEG-12-acetamido-GDM (1:5, drug to lipid ratio), the commercial product(Noxifil®, Schering-Plough), and a posaconazole solution containing 5%dimethyl sulfoxide and 10% Cremophor EL.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Embodiments of the present invention are described herein in the contextof PEG-lipid conjugates for liposomes and drug delivery. Those ofordinary skill in the art will realize that the following detaileddescription of the present invention is illustrative only and is notintended to be in any way limiting. Other embodiments of the presentinvention will readily suggest themselves to such skilled persons havingthe benefit of this disclosure. Reference will now be made in detail toimplementation of the present invention.

In the interest of clarity, not all of the routine features of theimplementations herein are described. It will be appreciated that in thedevelopment of such actual implementation, numerousimplementation-specific details must be made in order to achieve thedeveloper's specific goals, and that these specific goals will vary.Though such implementation might be complex, it will still be a routineexercise of engineering.

Herein, we describe biologically degradable linear PEG analogs carryingdiacylglycerol lipid groups. These new molecules belong to a classreferred to as diacylglycerol-polyethyleneglycols (DAG-PEGs). DAG-PEGshaving certain properties form liposomes spontaneously upon mixing withan aqueous solution. Briefly, DAG-PEGs or lipid mixtures includingDAG-PEGs must have packing parameters that allow liposome formation.Generally, P_(a) is preferably between about 0.84 and 0.88, and P_(v) ispreferably between about 0.88 and 0.93. Also, the DAG-PEGs or lipidmixtures including DAG-PEGs must have a melting point below thetemperature of liposome formation. If a DAG-PEG meets these conditions,it will form liposomes spontaneously (without external energy inputs).Spontaneous liposomes and DAG-PEGs are described in U.S. Pat. No.6,6610,322, which is hereby incorporated by reference. The particularDAG-PEGs described in U.S. Pat. No. 6,6610,322 utilized a single oxy(alternatively called oxyl) linkage between the PEG and the glycerolbackbone.

The present invention describes new linking chemical groups that can beselected to optimize and improve DAG-PEG liposome formulations.Selecting an appropriate linker between PEG and diacylglycerol can beimportant for several reasons, as described below.

Since a drug is a xenobiotic, the normal human body doesn't need it.Ideally, a drug should reach the site of action intact, cure thedisease, and leave the body after it completes its mission. However,drug developers often face the dilemma that a potential drug is eithermetabolized or excreted from the body too fast, so that the drug can notreach its site of action and achieve its therapeutic effect, or tooslow, so that it stays in the body for a long time causing side effects.An object of this invention is to develop PEG-lipids with unique linkersto help drugs to achieve therapeutic goals.

Liposomes can help drugs to penetrate cell membranes and to reach thesite of action. They can also confer an advantage by reducing specifictoxicities of some drugs, for example by reducing direct exposure of thedrug to organs or tissues susceptible to damage. Further they canimprove pharmacokinetics of a drug by delaying drug breakdown andclearance, serving as a sustained release drug depot, and also bytargeting sites of disease. Liposomes may be delivered topically,orally, sublingually or by intravenous administration. Thus, liposomeformulations must be optimized for each drug, disease, or route ofadministration. It is another object of this invention to provide newlipids to expand the range of possible liposome formulations.

Xenobiotics follow metabolic processes to be removed from the body. Thisprocess most commonly involves cytochrome P450 enzymes. These enzymesare a super family of proteins found in all living organisms. In humans,as well as all other mammalian species, this enzyme system is foundprincipally in the liver but exists in all other organs and tissues.These enzymes catalyze the following reactions: aromatic hydroxylation;aliphatic hydroxylation; N-, O-, and S-dealkylation; N-hydroxylation;N-oxidation; sulfoxidation and deamination. Of particular importance tothe present invention are the breakdown processes that the vesiclesformed from news lipids, and the new lipids themselves, are expected toundergo. Methoxyl and methylamine groups are expected to undergodemethylation. Amines are expected to undergo N-oxidation ordeamination. Sulfur bonds are expected to undergo S-oxidation. Estersand amides are expected to undergo hydrolysis. Since different organsand tissues have differing abilities to perform these differentreactions, it is a further objective of the present invention to providelinkers with optimal degradation properties.

Similarly, different microenvironments within the body favor differentbreakdown processes. For example, acidic gastric fluids favors breakdownof thiol linkages. Therefore, it is still another object of thisinvention to provide new molecules for designing drug deliveryformulations for diverse physiological microenvironments.

Of the two linked DAG-PEG components, diacylglycerol is digestible byhumans while PEG is not. However, as mentioned earlier, PEG is readilyexcreted. Breaking the linkage between the two components may result inincreased clearance for both. In is therefore an object of the inventionto optimize clearance rates of lipid vesicles and lipids used for drugdelivery.

Bilayer rigidity of liposomal drug formulations can be important toretain the drug within the liposome and to maintain liposomal integrityagainst clearance mechanisms of the body. Such rigidity can bemanipulated by adding sterols, other excipients and/or by selecting acylchains, though these methods result in a requirement for higher energyinput during liposome formation. The present invention provides anothermeans of controlling bilayer rigidity by selecting appropriate linkers.For example, due to the double bond character of the N, O or S withneighboring molecular bonds in some of these linkers, the barrier forrotation is very high resulting in more rigid liposomes. It is anotherobject of the invention to provide new materials and methods to controlrigidity of liposome membranes.

When using the lipids of the present invention to form spontaneousliposomes, the lipid mixtures used must fall within certain packingparameters. Since packing parameters of the DAG-PEGs depend on both thePEG portion and the diacylglycerol portion, breaking the link betweenthe two results in a change of overall packing parameters. Such aphenomena can be used to control liposomal breakdown and drug release.It is therefore another object of the invention to control liposomebreakdown and drug release by providing new DAG-PEGs with desirabledegradation properties.

Furthermore, these new linking chemical groups in the PEG-lipids havelarger polar surface areas than those having a single oxy linker. Forsome amphiphatic drugs or other compounds, this provides a betterenvironment for the drug or other compounds to partition into the lipidbilayer of the vesicle.

Narrow molecular weight distribution of drug delivery polymers iscrucially important for biomedical application, especially if used forintravenous injections. For instance, PEG-8 Caprylic/Capric Glyceridesare mixtures of monoesters, diesters, and triesters of glycerol andmonoesters and diesters of polyethylene glycols with a mean relativemolecular weight between 200 and 400. Partially due to allergicreactions observed in animals, the application of PEG-8 CCG for manywater-insoluble drugs was restricted and a dose limit of approximately6% of PEG-8 CCG was posted for human oral drug formulations.(http://www.accessdata.fda.gov/scripts/cder/iig/getiigWEB.cfm)

In one aspect, the present invention employs a new platform known asclick chemistry. [16. Binder, W. H.; Kluger, C. Macromolecules, 37(2004), 9321-9330; Díaz, D. D.; Punna, S.; Holzer, P.; McPherson, A. K.;Sharpless, K. B.; Fokin, V. V.; Finn, M. G. J. Polym. Sci., Part A:Polym. Chem. 42, (2004), 4392-4403; Helms, B.; Mynar, J. L.; Hawker, C.J.; Frechet, J. M. J. J. Am. Chem. Soc., 126 (2004), 15020-15021] Unlikefree radical polymerization, the molecular weight distributions may benarrowly controlled, typically within 10% of the targeted PEG molecularweight. Mono-distribution was achieved with purified PEGs. Thewell-defined PEG-diacylglycerol lipids prepared using this advancedtechnology may include all the various functional linker groupsdescribed herein.

A variety of new DAG-PEG lipids were synthesized and tested. The generalstructure of the new DAG-PEGs is

where R₁ is preferably either —OH or —OCH3; R₂ and R₃ are fatty acidsincluding and not limited to laurate, oleate, myristate, palmitate,stearate and linoleate; and X represents a single linker or replicatelinkers or combination of 2 or more listed linkers in between the lipidand PEG. Typically the R₂ and R₃ are the same, though they can also bedifferent. If R₂ is located at C1 position, R₃ can be located at eitherC2 or C3 position of the glycerol. The general structure is meant toinclude all racemers and structural isomers of the structure, as theycan be functionally equivalent.

Though R₁ is either —OH or —OCH₃ in the specific DAG-PEGs synthesizedand described herein, in practice R₁ has a negligible effect on theoverall DAG-PEG molecule and on liposome formation. Therefore, theinvention includes DAG-PEGs where R₁ is selected from a wide variety ofchemical moieties. Such moieties preferably have a molecular weight ofless than 215, and more preferably a molecular weight of less than 45.Such moieties include —NH₂, —COOH, —OCH₂CH₃, —OCH₂CH₂OH, —COCH═CH₂,—OCH₂CH₂NH₂, —OSO₂CH₃, —OCH₂C₆H₆, —OCH₂COCH₂CH₂COONC₄H₄O₂, —CH₂CH₂═CH₂,and —OC₆H₆. Also R₁ may be a functional group that facilitates linkingtherapeutic or targeting agents to the surface of a liposome. Aminoalkyl esters, maleimide, diglycidyl ether, maleinimido propionate,methylcarbamate, tosylhydrazone salts, azide, propargyl-amine, propargylalcohol, NHS esters (e.g., propargyl NHS ester, sulfo-NHS-LC-biotin, orNHS carbonate), hydrazide, succinimidyl ester, succinimidyl tartrate,succinimidyl succinate, and toluenesulfonate salt are useful for suchlinking. Linked therapeutic and targeting agents may include Fabfragments, cell surface binding agents, and the like. Additionally, R1may include functional cell-targeting ligands such as folate,transferrin and molecules such as monoclonal antibodies, ligands forcellular receptors or specific peptide sequences can be attached to theliposomal surface to provide specific binding sites. R1 can includeeither negatively or positively charged head-groups such asdecanolamine, octadecylolamine, octanolamine, butanolamine,dodecanolamine, hexanolamine, tetradecanolamine, hexadecanolamine,oleylamine, decanoltrimethylaminium, octadecyloltrimethylaminium,octanoltrimethylaminium, butanoltrimethylaminium,dodecanoltrimethylaminium, hexanoltrimethylaminium,tetradecanoltrimethylaminium, hexadecanoltrimethylaminium,oleyltrimethylaminium, for example.

Table 1 describes the linkers (“X”) used in the invention. Each of thelinkers provides unique advantages for liposome formation and drugdelivery. The structures shown in the table were mainly named byChemDraw. In the event of minor variations of chemical names, thestructures shown are meant to be controlling.

TABLE 1 Linkers No Symbol X 1 N₁

2 N₂

3 N₃

4 N₄

5 N₅

6 S₁

7 S₂

8 S₃

9 S₄

10 Ac₁

11 Ac₂

12 Ac₃

13 N₆

14 N₇

15 N₈

16 S₅

17 S₆

18 S₇

19 S₈

20 S₉

21 S₁₀

22 Ac₄

The invention can be practiced using a wide variety of fatty acids (R₂and R₃). Table 2 lists some saturated lipids for use in the invention.Table 3 lists some unsaturated lipids for use in the invention.

TABLE 2 Saturated lipids for use in the invention: Melting commonChemical point name IUPAC name structure Abbr. (° C.) Butyric Butanoicacid CH₃(CH₂)₂COOH C4:0 −8  Caproic Hexanoic acid CH₃(CH₂)₄COOH C6:0 −3 Caprylic Octanoic acid CH₃(CH₂)₆COOH C8:0 16-17 Capric Decanoic acidCH₃(CH₂)₈COOH C10:0 31  Lauric Dodecanoic acid CH₃(CH₂)₁₀COOH C12:044-46 Myristic Tetradecanoic acid CH₃(CH₂)₁₂COOH C14:0 58.8 PalmiticHexadecanoic acid CH₃(CH₂)₁₄COOH C16:0 63-64 Stearic Octadecanoic acidCH₃(CH₂)₁₆COOH C18:0 69.9 Arachidic Eicosanoic acid CH₃(CH₂)₁₈COOH C20:075.5 Behenic Docosanoic acid CH₃(CH₂)₂₀COOH C22:0 74-78

TABLE 3 Unsaturated lipids Δ^(x) Location of # carbon/ Name Chemicalstructure double bond double bonds Myristoleic acid CH₃(CH₂)₃CH═CH(CH₂)₇COOH cis-Δ⁹ 14:1 Palmitoleic acid CH₃(CH₂)₅ CH═CH(CH₂)₇COOHcis-Δ⁹ 16:1 Oleic acid CH₃(CH₂)₇ CH═CH(CH₂)₇COOH cis-Δ⁹ 18:1 Linoleicacid CH₃(CH₂)₄ CH═CHCH₂ CH═CH(CH₂)₇COOH cis, cis-Δ⁹, Δ¹² 18:2α-Linolenic acid CH₃CH₂ CH═CHCH₂ CH═CHCH₂ CH═CH(CH₂)₇ cis, cis, cis-Δ⁹,Δ¹², Δ¹⁵ 18:3 COOH Arachidonic acid CH₃(CH₂)₄ CH═CHCH₂ CH═CHCH₂ CH═CHCH₂cis, cis, cis, cis- Δ⁵Δ⁸, Δ¹¹, Δ¹⁴ 20:4 CH═CH(CH₂)₃COOH ^(NIST) Erucicacid CH₃(CH₂)₇ CH═CH(CH₂)₁₁COOH cis-Δ¹³ 22:1

A number of new DAG-PEGs were synthesized and tested. Most of thecombinations of PEG chains and fatty acids were chosen because it wasknown or expected that such DAG-PEGs with oxy linkages would havepacking parameters and melting points favorable for the formation ofliposomes. The new DAG-PEGs are shown in Table 4.

TABLE 4 Characterization of Representative New DAG-PEG-Lipids Meltingpoint Spontaneous Spontaneous Linker Lipid (° C.) P_(a) P_(v) Liposomeat 20° C. Liposome at 37° C. N₁ PEG-12-N₁-GDO Fluid @ 25 0.844 0.909 YesYes N₂ PEG-23-N₂-GDO Fluid @ 25 0.859 0.890 Yes Yes N₃ PEG-18-N₃-GDOFluid @ 25 0.869 0.903 Yes Yes N₄ PEG-23-N₄-GDO Fluid @ 25 0.847 0.892Yes Yes S₁ PEG-8-S₁-GDO Fluid @ 25 0.830 0.925 Yes Yes S₂ PEG-18-S₂-GDOFluid @ 25 0.852 0.890 Yes Yes S₃ PEG-12-S₃-GDO Fluid @ 25 0.847 0.892Yes Yes Ac₁ PEG-18-Ac₁-GDO Fluid @ 25 0.843 0.886 Yes Yes Ac₂PEG-12-Ac₂-GDO Fluid @ 25 0.848 0.883 Yes Yes N₁ PEG-12-N₁-GDM Fluid @25 0.856 0.908 Yes Yes N₁ PEG-12-N₁-GDLO Fluid @ 25 0.850 0.924 Yes YesS₃ PEG-12-S₃-GDM Fluid @ 25 0.854 0.886 Yes Yes S₃ PEG-12-S₃-GDLO Fluid@ 25 0.855 0.899 Yes Yes Ac₂ PEG-12-Ac₂-GDM Fluid @ 25 0.846 0.884 YesYes Ac₂ PEG-12-Ac₂-GDLO Fluid @ 25 0.854 0.885 Yes Yes N₁ PEG-23-N₁-GDLFluid @ 25 0.866 0.919 Yes Yes N₁ PEG-12-N₁-GDP Fluid @ 25 0.840 0.914Yes Yes Ac₂ PEG-23-Ac₂-GDL Fluid @ 25 0.842 0.872 Yes Yes Ac₂PEG-12-Ac₂-GDP Fluid @ 25 0.866 0.884 Yes Yes

In Table 4 the symbols for the linkers are from Table 1. GDO meansglycerol dioleate, GDM means glycerol dimyristate, GDLO means glyceroldilinoleate, GDL means glycerol dilaurate, and GDP means glyceroldipalmitate. The numeral after the PEG means the number of subunits inthe PEG chain. For example, PEG-12 refers to a PEG chain having 12subunits.

Packing parameters P_(a) and P_(v) were calculated using the followingequation (Lasic D. D. “Liposomes: From Physics to Application,”Elsevier, Amsterdam (1993), 49-51; Keller, C. B., Chapter 12, “Handbookof Cosmetic Science & Technology, edited Paye M., Barel, A. O., Maibach,H. I., Taylor & Francis, New York (2006), 165-174:

$P_{a} = {{\eta\frac{\;{F_{a}\lambda}}{T_{v}}\mspace{14mu}{and}\mspace{14mu} P_{v}} = {\eta\;\frac{F_{v}}{T_{v}}}}$where η is a fraction factor which is related to the purity andhomogeneity of the PEG-lipid (η=η₁+η₂+ . . . =1). F_(a) is the polarsurface area of the lipid, λ is length of lipophilic portion, F_(v) andT_(v) are the volumes of lipophilic portion and the whole molecule.ChemDraw (CambridgeSoft) and ChemSketch (Advanced Chemistry Development,Inc) were used to perform the calculations with the experimental data.

As described in U.S. Pat. No. 6,6610,322, combinations of lipids may beused to form liposomes if the packing parameters of the lipidcombination as a whole are within favorable ranges. Thus, even PEG-lipidconjugates of the present invention which by themselves have packingparameters outside the desired range may be incorporated into liposomesby combining them with other lipids, or with other compounds such assterols that affect overall packing parameters.

The use of new linkers between the C3 position of glycerol and the PEGchain did not significantly change the packing parameters or meltingpoints of the new DAG-PEGS as compared to DAG-PEGs with oxy linkages.However due to the double bond character of the N, O or S withneighboring molecular bond in some of these linkers, the barrier forrotation is very high. The rate of conformational changes can be furtherlowered by involvement of these molecules in hydrogen bonding withneighboring molecules or fragments of the same molecules, giving rise torigid conformers which may not be favorable for the formation ofliposomes at an ambient temperature. In such case, longer formation timeor elevated temperatures to increase the rate of liposome formations arenecessary.

Liposomes incorporating PEG-lipid conjugates of the present inventionmay be used to encapsulate and deliver a wide variety of active agentssince the liposomes include an enclosed aqueous space, a hydrophobicregion in the bilayer, and sites for covalent attachment. Such activeagents may include proteins, peptides, nucleic acids, antineoplasticagents, anti-inflammatories, anti-infectives, gastrointestinal agents,biological and immunological agents, dermatologic agents, ophthalmic andotic agents, diagnostic aids, nutrients and nutritional agents,hematological agents, endocrine and metabolic agents, cardivasculars,renal and genitourinary agents, central nervous system agents.

The syntheses used in this invention to formdiacylglycerol-polyethyleneglycols generally utilizes the reaction ofthe PEG polymer with a linker that is reactive with hydroxyl groups,typically anhydrides, acid chlorides, chloroformates and carbonates,aldehyde, esters, amides etc ore more efficient functional groups forthe conjugation. Preferred end groups include maleimide, vinyl sulfones,pyridyl disulfide, amine, carboxylic acids and NHS esters.

The DAG-PEGs and linkers disclosed herein can be considered as a “toolkit” to aid in the design of lipsomes. The linkers described offer avariety of options in terms of size, potential binding sites, hydrogenbonding, polarity, and breakdown reactions. By selecting particularlinkers, incorporation and retention of encapsulated agents can beimproved. The lipophobic portion of amphiphatic molecules may bestabilized by selecting the appropriate linker. For example, linkers Ac₄and N₁ differ greatly in their potentials to form hydrogen bonds.

The effective combination of functional head-groups, lipid chains andlinker groups in membrane components can achieve controlled stability ofthe liposomal membrane and selective release of encapsulated materialunder specific environmental conditions. Degradation of the DAG-PEGs,with concurrent release of encapsulated compounds, can thus becontrolled by selecting appropriate linkers. For instance, pH dependenthydrolysis of the non-charged cleavable linkers containing C—N or C—Obonds and the thiolysis of the linkers containing disulfide bonds thatare integrated in the membrane occur. In some cases, release agents mayexpedite such breakdown. Examples of linkers whose breakdown rate isincreased by release agents include: S₈, S₉, and S₄, which break downfaster under hypoxic conditions; S₂, S₁₀, and N₆, which break down athigh pH; and S₈ and S₉, which are sensitive to ultraviolet light.Catalysts may also increase the rate of breakdown of particular linkers.For example, S₈ and S₉ break down faster in the presence of H⁺. Heat mayincrease the rate of acid-catalyzed linker degradation. FIG. 1demonstrates enhanced breakdown of linker S₂ at low pH. It is worthnoting that degradation may not be always a “breakdown,” it is possibleto form a secondary product under some conditions.

Even though degradation of some of the linkers occurs in the presence ofrelease agents, the new DAG-PEGs of the invention are generally muchmore stable than phospholipids. For example, phospholipids degrade in amatter of days at room temperature while the new lipids are stable forlong periods of time at room temperature.

Synthesis of the new lipids may be controlled so that there is a singlelinker in each DAG-PEG molecule. In some situations, however, it may beuseful to have multiple copies of the same linker, or combinations ofdifferent linkers in a single molecule.

The liposomes of the present invention may be used for manyapplications. Formulation and delivery of pharmaceutical and cosmeticagents have been described. Additionally, the DAG-PEGs of the presentinvention may be used in other contexts where liposomes are advantages,for example industrial and/or cleaning processes.

In one aspect the invention includes a compound represented by theformula

where R₁ preferably has a molecular weight of less than about 215; whereR₂ and R₃ are alkyl groups having between 4 and 22 carbons; and where Xis one or more linkers selected from the group consisting of amino,succinylamino, acetamido, aminopentanamido, aminoacetyl, thiopropanoayl,N-(mercaptomethyl)propionamido, mercaptopropylthio)-propanoyl,(1,2-dihydroxy-3-mercaptopropylthio)propanoyl, succinyl, acetyl,oxopentanoyl, carbamoyl, aminoalkyl, glutaramido, aminoethanethiol,mercaptopropanol, (hydroxypropylthio)propanoayl,3-((2-propionamidoethyl)disulfanyl)propanoayl,(((acetamidoethyl)disulfanyl)propanoyloxy)glutaramido,aminoethanethioate, and 2-hydroxyacetic proprionic anhydride. Morepreferably R₁ has a molecular weight of less than about 45. R₁ may beeither —OH or —OCH₃. R₂ and R₃ may preferably be selected from the groupconsisting of oleate, myristate, linoleate and palmitate. The PEG chainmay consist of between about 6 and 45 subunits. More preferably the PEGchain consists of between about 8 and 23 subunits. Still more preferablythe PEG chain consists of between about 12 and 23 subunits. The compoundis useful for applications other than liposomes, e.g., as a solvent.

In another aspect the invention includes a liposome comprising acompound represented by the formula

where R₁ preferably has a molecular weight of less than about 215;where R₂ and R₃ are alkyl groups having between 4 and 22 carbons; andwhere X is selected from the group consisting of amino, succinylamino,acetamido, aminopentanamido, aminoacetyl, thiopropanoayl,N-(mercaptomethyl)propionamido, mercaptopropylthio)-propanoyl,(1,2-dihydroxy-3-mercaptopropylthio)propanoyl, succinyl, acetyl,oxopentanoyl, carbamoyl, aminoalkyl, glutaramido, aminoethanethiol,mercaptopropanol, (hydroxypropylthio)propanoayl,3-((2-propionamidoethyl)disulfanyl)propanoayl,(((acetamidoethyl)disulfanyl)propanoyloxy)glutaramido,aminoethanethioate, and 2-hydroxyacetic proprionic anhydride. Morepreferably R₁ has a molecular weight of less than about 45. R₁ may beeither —OH or —OCH₃. R₂ and R₃ may be selected from the group consistingof oleate, myristate, linoleate and palmitate. It may be preferable tohave molecular weight oligomers of PEG greater than 400 Da (or >8subunits) since the lower molecular weight of PEG (i.e., <400 Da) havebeen shown to be degraded in vivo by alcohol dehydrogenase to toxicmetabolites [Newman, J. Johnson et al., “Poly(ethylene glycol) Chemistryand Biological Applications,” J. M. Harris and S. Zalipsky, Editors, ACSBooks, Washington, D.C. (1997), 45-57]. However, some PEG-6 DAG-PEGs areuseful for forming liposomes by themselves and in combination with otherDAG-PEGs. Therefore, the PEG chain may consist of between about 6 and 45subunits. More preferably the PEG chain may consist of between about 8and 23 subunits. Even more preferably the PEG chain consists of betweenabout 12 and 23 subunits. The liposome may comprise one or more activeagents. The active agent may be a tetrahydrofuran. The active agent maybe rifampicin.

In yet another aspect, the invention includes a method of preparing aliposome formulation of a therapeutic agent, the method comprisingdetermining a therapeutic target; determining a mode of administration;determining the physiological conditions the liposome will encounter inreaching the therapeutic target using the mode of administration;selecting a DAG-PEG lipid having a linker between the PEG chain and theglycerol backbone, where such selecting is informed by the physiologicalconditions; and combining the DAG-PEG lipid and the therapeutic agent ina liposome formulation. The linker is selected from the group consistingof amino, succinylamino, acetamido, aminopentanamido, aminoacetyl,thiopropanoayl, N-(mercaptomethyl)propionamido,mercaptopropylthio)-propanoyl,(1,2-dihydroxy-3-mercaptopropylthio)propanoyl, succinyl, acetyl,oxopentanoyl, carbamoyl, aminoalkyl, glutaramido, aminoethanethiol,mercaptopropanol, (hydroxypropylthio)propanoayl,3-((2-propionamidoethyl)disulfanyl)propanoayl,(((acetamidoethyl)disulfanyl)propanoyloxy)glutaramido,aminoethanethioate, and 2-hydroxyacetic proprionic anhydride.

In another aspect the invention includes a DAG-PEG having the generalstructure

where R₁ has a molecular weight of less than about 215; where R₂ and R₃are alkyl groups having between 4 and 22 carbons where X breaks done atan accelerated rate in the presence of a release agent selected from thegroup consisting of acid, hypoxia, light, and catalyst.

Another aspect of the invention includes a method of delivering acompound, where the method comprises preparing a liposome formulation ofthe compound, where the liposome comprises a DAG-PEG having a linkerselected from the group consisting of amino, succinylamino, acetamido,aminopentanamido, aminoacetyl, thiopropanoayl,N-(mercaptomethyl)propionamido, mercaptopropylthio)-propanoyl,(1,2-dihydroxy-3-mercaptopropylthio)propanoyl, succinyl, acetyl,oxopentanoyl, carbamoyl, aminoalkyl, glutaramido, aminoethanethiol,mercaptopropanol, (hydroxypropylthio)propanoayl,3-((2-propionamidoethyl)disulfanyl)propanoayl,(((acetamidoethyl)disulfanyl)propanoyloxy)glutaramido,aminoethanethioate, and 2-hydroxyacetic proprionic anhydride; locatingthe liposome at a desired site; and providing a release agent, where therelease agent causes the linker to degrade. The release agent may be anacid, light, hypoxia, or a catalyst.

EXAMPLES

Chemicals and Reagents: N,N′-dicyclohexylurea,N,N′-dicyclohexylcarbodiimide, DL 1,2-rac-dioleoylglycerol, sodiumcyanoborohydride, dimethyl sulfoxide (DMSO) and other chemicals wereobtained from Sigma-Aldrich (St. Louis, Mo., USA). Activated PEGpolymers including poly(ethylene glycol)₁₂-aldehyde, poly(ethyleneglycol)₁₂-succinyl-D,L-dithiothreitol, Poly(ethyleneglycol)₁₂-monomethyl ether succinate and 3-amino-1,2-rac-dioleoylglycerol were supplied by Novus PharmTech, Ltd (Nanjing, China). Bulkquantity of DL 1,2-rac-dioleoylglycerol is commercially available andcan be purchased from Spectrum Chemicals (Gardena, Calif., USA).

Example 1 Synthesis of PEG-amino (N₁)-GDO

3-amino-1,2-rac-dioleoyl glycerol (˜8 moles) and potassium hydroxide(KOH, 0.5 moles) were charged into a reactor. The reaction mixture washeated to a temperature of 120-130° C. under nitrogen for two hours,with ethylene oxide (in an amount calculated to result in polymers withan average chain length of eight subunits) added while held at atemperature of 120-130° C. The reaction was completed when free ethyleneoxide was not detectable. The resulting brine was continuouslyextracted, typically for 4 hours at 55° C. with ethyl acetate. Theproduct was dried out of the solvents under vacuo.

Example 2 Synthesis of PEG-12-Succinylamino (N₂)-GDO

A mixture of 3-amino-1,2-rac-dioleoyl glycerol (0.1 mol); poly(ethyleneglycol)₁₂-monomethyl ether succinate (0.1 mol);N,N′-dicyclohexylcarbodiimide (0.1 mol); and a catalytic amount of4-dimethylaminopyridine in anhydrous Dichloromethane (200 ml) wasstirred at room temperature for 24 h, after which theN,N′-dicyclohexylurea salts were precipitated and removed by filtration.The filtrates were evaporated under reduced pressure. The crude productwas purified by chromatography on a silica gel (G60, grade 60, mesh240-400) column. The column was eluted with a 1:1 (v/v) diethylether/acetone mixture. The first fraction eluted was unreacted reagentsand discarded. The peg-lipid peak at 220 nm (UV monitoring) wascollected. Free PEGs were washing out the column by 100% acetone. Theproduct was dried out of the solvents under vacuo.

Example 3 Synthesis of PEG-12-Acetamido (N₃)-GDO

3-amino-1,2-rac-dioleoyl glycerol was dissolved in ethanol/50 mM sodiumphosphate (1/1, v/v). The final concentration was 0.1 molar.Poly(ethylene glycol)₁₂-aldehyde (0.5 mol) were added to the solution,the pH was adjusted to 8.0, stirred at room temperature for 20 h. Theproduct was purified on a C8 column, using acetonitrile/10 mM ammoniumacetate, pH 6.6, then a linear gradient of methanol from 60 to 100% in15 min, followed by a 10 min isocratic step with 100% methanol as theelution system. Peaks were detected for collection at 220 nm. Thecollected fraction was then freeze-dried.

Example 4 Synthesis of PEG-12-Aminopentanamido (N₄)-GDO

3-amino-1,2-rac-dioleoyl glycerol was dissolved in ethanol/0.2M boratebuffer, pH 8.5, 1/1 (final concentration 0.5 g/mL), and a 5-fold excessof poly(ethylene glycol)₁₂ norleucine-N-hydroxysuccinimide (Sartore, L.,Caliceti, P., Schiavon, O., Monfardini, C., and Veronese, F. M.,Accurate evaluation method of the polymer content inmonomethoxypoly(ethylene glycol) modified proteins based on amino acidanalysis. Appl. Biochem. Biotechnot 31(1991), 213-222) was added whilestirring. The reaction was over after 6 h at room temperature, asconfirmed by analytical RP-HPLC. The product was purified on a C8column, using acetonitrile/50 mM ammonium acetate, pH 6.5, then a lineargradient of methanol from 60 to 100% in 15 min, followed by a 10 minisocratic step with 100% methanol as the elution system. Peaks weredetected for collection at 220 nm. The collected fraction was thenfreeze-dried.

Example 5 Synthesis of PEG-12-Aminoacetyl (N₅)-GDO

3-amino-1,2-rac-dioleoyl glycerol was dissolved in ethanol/50 mM sodiumphosphate (1/1, v/v). The final concentration was 0.1 molar.Poly(ethylene glycol)₁₂-aldehyde (0.5 mol) and sodium cyanoborohydride(0.2 mol) were added to the solution, the pH was adjusted to 8.0, andthe mixture was stirred at room temperature for 20 h. The product waspurified on a C8 column, using acetonitrile/10 mM ammonium acetate, pH6.6, as elution systems. Peaks were detected at 220 nm. A lineargradient of methanol from 60 to 100% in 15 min was used, followed by a10 min isocratic step with 100% methanol. The collected fraction wasthen freeze-dried.

Example 6 Synthesis of PEG-12-Thiopropanoyl (S₁)-GDO

mPEG 600 (0.5 moles) in 150 mL of DMF, was reacted with3-mercaptopropionic acid (10 mol, 20 equiv) in the presence2,T-azobisisobutyronitrile (1 mol equiv). The reaction mixture wasstirred at 65° C. for 24 h under argon atmosphere. The polymer wasprecipitated twice in a large excess of ether. The resulting whiteproduct was dissolved into methanol, and potassium hydroxide (1 molequiv) dissolved in water was added. The mixture was stirred forapproximately 4 h. Then, methanol was partially evaporated and dilutedwith water (100 mL) and extracted by dichloromethane (5×100 mL). Thecombined organic layer was dried over Na₂SO₄, filtered, and concentratedto 1/100 of the initial volume. The polymer was reprecipitated from anexcess volume of ether and freeze-dried from benzene to and transferredto the next step. The transferred resultant (0.1 mol), dioleoyl glycerol(0.1 mol), N,N′-dicyclohexylcarbodiimide (0.1 mol) and a catalyticamount of 4-dimethylaminopyridine in anhydrous Dichloromethane (200 ml)was stirred at room temperature for 24 h, after which theN,N′-dicyclohexylurea salts were precipitated and removed by filtration.The filtrates were evaporated under reduced pressure. The crude productwas purified by chromatography on a silica gel (G60, grade 60, mesh240-400) column. The column was eluted with a 1:1 (v/v) diethylether/acetone mixture. The first fraction eluted was unreacted reagentsand discarded. The peg-lipid peak at 220 nm (UV monitoring) wascollected. Free PEGs were washing out the column by 100% acetone. Theproduct was dried out of the solvents under vacuo.

Example 7 Synthesis of PEG-12-(mercaptomethyl)propionamido (S₂)-GDO

mPEG 600 (0.91 mmol) was freeze dried from benzene and mixed with thesolution a solution of DMF (50 mL) containing 2-aminoethanethiolhydrochloride (1.87 mol, 20 equiv) and 2,2′-azobisisobutyronitrile (1mol equiv). The reaction mixture was stirred at 65° C. for 24 h underargon atmosphere. The polymer was precipitated twice in a large excessof ether. The resulting white product was dissolved into methanol, and5.1 mg (1 equiv) of potassium hydroxide dissolved in water was added.The mixture was stirred for approximately 4 h. Then methanol waspartially evaporated and the mixture was diluted with water (30 mL) andextracted by dichloromethane (5×80 mL). The combined organic layer wasdried over Na₂SO₄, filtered, and concentrated to 1/100 of the initialvolume. The polymer was reprecipitated from an excess volume of etherand then freeze-dried from benzene and transferred to the next step. Thetransferred resultant (0.1 mol), dioleoyl glycerol (0.1 mol),N,N′-dicyclohexylcarbodiimide (0.1 mol) and a catalytic amount of4-dimethylaminopyridine in anhydrous Dichloromethane (200 ml) wasstirred at room temperature for 24 h, after which theN,N′-dicyclohexylurea salts were precipitated and removed by filtration.The filtrates were evaporated under reduced pressure. The crude productwas purified by chromatography on a silica gel (G60, grade 60, mesh240-400) column. The column was eluted with a 1:1 (v/v) diethylether/acetone mixture. The first fraction eluted was unreacted reagentsand discarded. The peg-lipid peak at 220 nm (UV monitoring) wascollected. Free PEGs were washed out the column by 100% acetone. Theproduct was dried out of the solvents under vacuo.

Example 8 Synthesis of PEG-12-2-(3-mercaptopropylthio)propanoyl (S₃)-GDO

mPEG-1,2-ethanedithiol-propanoate was prepared by a Michael typepolyaddition of mPEG 600 methyl acrylate with 1,2-ethanedithiol.1,2-ethanedithiol-(3.1313 mol) was dissolved in 300 mL of DMSO at roomtemperature. mPEG 600 methyl acrylate (3.1310 mol) was added to the DMSOsolution. Triethanolamine (0.04 mol) was added dropwise to the solution.The solution was stirred at room temperature for 72 h. The polymer wasprecipitated in ether and further purified by repeated precipitation.The precipitate was dried in a vacuum oven at 70° C. overnight andtransferred to the next step. mPEG-1,2-ethanedithiol-propanoate (0.5mol) and succinic dioleoylglycerol (0.5 mol) were dissolved in 100 mL ofdry DMSO and stirred at 60° C. for 72 h until all hydroxyl groups werereacted. The resulting PEG-S₃-lipid was isolated in ether and driedunder a high vacuum at 60° C. overnight. Crude product in brine wascontinuously extracted, typically for 4 hours at 55° C. with ethylacetate. The product was dried out of the solvents under vacuo.

Example 9 Synthesis ofPEG-12-2-(1,2-dihydroxy-3-mercaptopropylthio)propanoyl (S₄)-GDO

mPEG-D,L-dithiothreitol-propanoate was prepared by a Michael typepolyaddition of mPEG 600 methyl acrylate with D,L-dithiothreitol.1,2-ethanedithiol-(3.1313 mol) was dissolved in 300 mL of DMSO at roomtemperature. mPEG 600 methyl acrylate (3.1310 mol) was added to the DMSOsolution. Triethanolamine (0.04 mol) was added dropwise to the solution.The solution was stirred at room temperature for 72 h. The polymer wasprecipitated in ether and further purified by repeated precipitation.The precipitate was dried in a vacuum oven at 70° C. overnight andtransferred to the next step. mPEG-1,2-D,L-dithiothreitol-propanoate(0.5 mol) and succinic dioleoylglycerol (0.5 mol) were dissolved in 100mL of dry DMSO and stirred at 60° C. for 72 h until all hydroxyl groupswere reacted. The resulting PEG-S₃-lipid was isolated in ether and driedunder a high vacuum at 60° C. overnight. Crude product in brine wascontinuously extracted, typically for 4 hours at 55° C. with ethylacetate. The product was dried out of the solvents under vacuo.

Example 10 Synthesis of PEG-12-Succinyl (Ac₁)-GDO

Dioleoyl glycerol (0.1 mol), poly(ethylene glycol)₁₂-monomethyl ethersuccinate (0.1 mol), N,N′-dicyclohexylcarbodiimide (0.1 mol) and acatalytic amount of 4-dimethylaminopyridine in anhydrous Dichloromethane(200 ml) was stirred at room temperature for 24 h, after which theN,N′-dicyclohexylurea salts were precipitated and removed by filtration.The filtrates were evaporated under reduced pressure. The crude productwas purified by chromatography on a silica gel (G60, grade 60, mesh240-400) column. The column was eluted with a 1:1 (v/v) diethylether/acetone mixture. The first eluted fraction was unreacted reagents,and was discarded. The PEG-lipid peak at 220 nm (UV monitoring) wascollected. Free PEGS were washed out the column by 100% acetone. Theproduct was dried out of the solvents under vacuo.

Example 11 Synthesis of PEG-12-Acetyl (Ac₂)-GDO

Dioleoyl glycerol (0.1 mol), monocarboxylpoly(ethyleneglycol)₁₂-monomethyl ether (0.1 mol), N,N′-dicyclohexylcarbodiimide (0.1mol) and a catalytic amount of 4-dimethylaminopyridine in anhydrousDichloromethane (200 ml) was stirred at room temperature for 24 h, afterwhich the N,N′-dicyclohexylurea salts were precipitated and removed byfiltration. The filtrates were evaporated under reduced pressure. Thecrude product was purified by chromatography on a silica gel (G60, grade60, mesh 240-400) column. The column was eluted with a 1:1 (v/v) diethylether/acetone mixture. The first eluted fraction was unreacted reagents,and was discarded. The peg-lipid peak at 220 nm. (UV monitoring) wascollected. Free PEGs were washed out the column by 100% acetone. Theproduct was dried out of the solvents under vacuo.

Example 12 Synthesis of PEG-12-5-oxopentanoyl (Ac₃)-GDO

Dioleoyl glycerol (0.1 mol), mono-5-oxopentanoyl poly(ethyleneglycol)₁₂-monomethyl ether (0.1 mol), N,N′-dicyclohexylcarbodiimide (0.1mol) and a catalytic amount of 4-dimethylaminopyridine in anhydrousDichloromethane (200 ml) were stirred at room temperature for 24 h,after which the N,N′-dicyclohexylurea salts were precipitated andremoved by filtration. The filtrates were evaporated under reducedpressure. The crude product was purified by chromatography on a silicagel (G60, grade 60, mesh 240-400) column. The column was eluted with a1:1 (v/v) diethyl ether/acetone mixture. The first fraction eluted wasunreacted reagents, and was discarded. The peg-lipid peak at 220 nm (UVmonitoring) was collected. Free PEGs were washed out the column by 100%acetone. The product was dried out of the solvents under vacuo.

Example 13 Synthesis of PEG-12-hydroxyacetic propionic anhydrido(Ac₄)-GDO

Dioleoyl glycerol (0.1 mol), 2-hydroxyacetic 2-(mPEG-12)-aceticanhydride (0.1 mol), N,N′-dicyclohexylcarbodiimide (0.1 mol) and acatalytic amount of 4-dimethylaminopyridine in anhydrous Dichloromethane(200 ml) were stirred at room temperature for 24 h, after which theN,N′-dicyclohexylurea salts were precipitated and removed by filtration.The filtrates were evaporated under reduced pressure. The crude productwas purified by chromatography on a silica gel (G60, grade 60, mesh240-400) column. The column was eluted with a 1:1 (v/v) diethylether/acetone mixture. The first fraction eluted was unreacted reagents,and was discarded. The peg-lipid peak at 220 nm (UV monitoring) wascollected. Free PEGs were washed out the column by 100% acetone. Theproduct was dried out of the solvents under vacuo.

Example 14 Synthesis of PEG-12-Carbamoyl (N₆)-GDO

3-hydroxyl-1,2-rac-dioleoyl glycerol was dissolved in DMF. The finalconcentration was 0.1 molar. Poly(ethylene glycol)₁₂-aminoacetic acid(0.1 mol), and a catalytic amount of 4-dimethylaminopyridine inanhydrous DMF were added to the solution and stirred at room temperaturefor 24 h. The product was purified on a C8 column, using acetonitrile/10mM ammonium acetate, pH 6.6, a linear gradient of methanol from 60 to100% in 15 min, followed by a 10 min isocratic step with 100% methanolas the elution system. Peaks were detected at 220 nm. And collected. Thecollected fraction was then freeze-dried.

Example 15 Synthesis of PEG-12-N₇-GDO

The synthesis was same as for PEG-12-Amino (N₁)-GDO.

Example 16 Synthesis of PEG-12-glutaramido (N₈)-GDO

Methoxy-PEG 600 N-succinimidyl ester (0.1 mol) and 3 mL of triethylaminewere added to a solution of 3-amino-1,2-rac-dioleoyl glycerol (0.1 mol)in 250 mL of CHCl₃, and the reaction mixture was stirred for 2 hours at15° C. The solvent was evaporated, and the residue was dissolved in 100mL of CHCl₃ and purified by chromatography on 200 g of silica gel.Elution was with with 250 mL of CHCl₃/MeOH, 90/10 (v/v) and 250 mL ofCHCl₃/MeOH, 70/30 (v/v).

Example 17 Synthesis of PEG-12-mercaptopropanol (S₅)-GDO

Allyl-PEG 600 (0.1 mol) was mixed with the solution in DMF (500 mL)containing 3-ethanethiol-dl-1,2-dioleoyl glycerol (0.1 mol, 1 equiv) and2,2′-azobisisobutyronitrile (0.1 mol, 1 equiv). The reaction mixture wasstirred at 65° C. for 24 h under argon atmosphere. The polymer wasprecipitated twice in a large excess of ether. The resulting whiteproduct was dissolved into methanol, and 5.1 mg (1 equiv) of potassiumhydroxide dissolved in water was added. The mixture was stirred forapproximately 4 h, then diluted with water (100 mL) and extracted bydichloromethane (5×150 mL). The combined organic layer was dried overNa₂SO₄, filtered, and concentrated to 1/100 of the initial volume. Thepolymer was reprecipitated from an excess volume of ether andfreeze-dried from benzene.

Example 18 Synthesis of PEG-12-(hydroxypropylthio)propanoayl (S₆)-GDO

Allyl-PEG 600 (0.1 mol) was mixed with the solution in DMF (500 mL)containing 3-mercaptopropionic acid-dl-1,2-dioleoyl glycerol (0.1 mol, 1equiv) and 2,2′-azobisisobutyronitrile (0.1 mol, 1 equiv). The reactionmixture was stirred at 65° C. for 24 h under argon atmosphere. Thepolymer was precipitated twice in a large excess of ether. The resultingwhite product was dissolved into methanol, and 5.1 mg (1 equiv) ofpotassium hydroxide dissolved in water was added. The mixture wasstirred for approximately 4 h, then diluted with water (100 mL), andextracted by dichloromethane (5×150 mL). The combined organic layer wasdried over Na₂SO₄, filtered, and concentrated to 1/100 of the initialvolume. The polymer was reprecipitated from an excess volume of etherand freeze-dried from benzene to yield a yellowish liquid.

Example 19 Synthesis of PEG-12-mercaptopropanoayl (S₇)-GDO

Poly(ethylene glycol) 750 methacrylate (0.1 mol) was mixed with thesolution in DMF (500 mL) containing 3-ethanethiol-dl-1,2-dioleoylglycerol (0.1 mol, 1 equiv) and 2,2′-azobisisobutyronitrile (0.1 mol, 1equiv). The reaction mixture was stirred at 65° C. for 24 h under argonatmosphere. The polymer was precipitated twice in a large excess ofether. The resulting white product was dissolved into methanol, and 5.1mg (1 equiv) of potassium hydroxide dissolved in water was added. Themixture was stirred for approximately 4 h, then diluted with water (100mL), and extracted by dichloromethane (5×150 mL). The combined organiclayer was dried over Na₂SO₄, filtered, and concentrated to 1/100 of theinitial volume. The polymer was reprecipitated from an excess volume ofether and freeze-dried from benzene to yield a yellowish liquid.

Example 20 Synthesis of PEG12-3-((2-propionamidoethyl)disulfanyl)propanoayl (S₈)-GDO

A solution of PEG-12-3-((2-propionamidoethyl)disulfanyl)propanoic acid(0.2 mol) in 250 mL of dry pyridine (pre-dried by placing 4 Angstrommolecular sieves into reagent grade pyridine, shaking and letting standat least overnight) was stirred at room temperature under an argonatmosphere. A solution of 3-amino-1,2-rac-dioleoyl glycerol (0.2 mmol)in 50 mL of dry Pyridine was added dropwise over a period of 30 min. Thesolution was stirred for an additional 60 min, 10 mL of acetic acid wasadded, and the solvent was evaporated. Residual acetic acid wasazeotroped with toluene. The residue was dissolved in 100 mL of CHCl₃and subjected to chromatography on 500 g of silica gel. The product waseluted by first applying 500 mL of CHCl₃/MeOH/AcOH=90/10/0.1 and then600 mL of 70/30/0.5. Fractions containing the product were pooled,evaporated, and azeotroped with toluene. The residue was again dissolvedin CHCl₃, filtered, and evaporated to yield a yellowish liquid.

Example 21 Synthesis of PEG12-N¹-(3-((2-acetamidoethyl)disulfanyl)propanoyloxy)-glutar amido(S₉)-GDO

A solution of PEG-12-2,5-dioxopyrrolidin-1-yl3-((2-acetamidoethyl)disulfanyl)-propanoate (0.2 mol) in 250 mL of dryPyridine (pre-dried by placing 4 Angstrom molecular sieves into reagentgrade Pyridine, shaking and letting stand at least overnight) wasstirred at room temperature under an argon atmosphere. A solution of3-amino-1,2-rac-dioleoyl glycerol (0.2 mmol) in 50 mL of dry pyridinewas added dropwise over a period of 30 min. The solution was stirred foran additional 60 min, 10 mL of acetic acid was added, and the solventwas evaporated. Residual acetic acid was azeotroped with toluene. Theresidue was dissolved in 100 mL of CHCl₃ and subjected to chromatographyon 500 g of silica gel. The product was eluted by first applying 500 mLof CHCl₃/MeOH/AcOH=90/10/0.1 and then 600 mL of 70/30/0.5. Fractionscontaining the product were pooled, evaporated, and azeotroped withtoluene. The residue was again dissolved in CHCl₃, filtered, andevaporated to yield a yellowish liquid.

Example 22 Synthesis of PEG 12-aminoethanethioayl (S₁₀)-GDO

Allyl-PEG 600 (0.1 mol) was mixed with the solution in DMF (500 mL)containing dl-1,2-dioleoyl glycerol-3-aminoethanethioic S-acid (0.1 mol,1 equiv) and 2,2′-azobisisobutyronitrile (0.1 mol, 1 equiv). Thereaction mixture was stirred at 65° C. for 24 h under argon atmosphere.The polymer was precipitated twice in a large excess of ether. Theresulting white product was dissolved into methanol, and 5.1 mg (1equiv) of potassium hydroxide dissolved in water was added. The mixturewas stirred for approximately 4 h, then diluted with water (100 mL), andextracted by dichloromethane (5×150 mL). The combined organic layer wasdried over Na₂SO₄, filtered, and concentrated to 1/100 of the initialvolume. The polymer was reprecipitated from an excess volume of etherand freeze-dried from benzene.

Example 23 Synthesis of PEG-12-hydroxyacetic propionic anhydrido(Ac₄)-GDO

Dioleoyl glycerol (0.1 mol), 2-hydroxyacetic 2-(mPEG-12)-aceticanhydride (0.1 mol), N,N′-dicyclohexylcarbodiimide (0.1 mol) and acatalytic amount of 4-dimethylaminopyridine in anhydrous Dichloromethane(200 ml) were stirred at room temperature for 24 h, after which theN,N′-dicyclohexylurea salts were precipitated and removed by filtration.The filtrates were evaporated under reduced pressure. The crude productwas purified by chromatography on a silica gel (G60, grade 60, mesh240-400) column. The column was eluted with a 1:1 (v/v) diethylether/acetone mixture. The first fraction eluted was unreacted reagents,and was discarded. The peg-lipid peak at 220 nm (UV monitoring) werecollected. Free PEGs were washing out the column by 100% acetone. Theproduct was dried out of the solvents under vacuo.

Example 24 In Vitro Stability of Selective Linkers

Three DAG-PEGs with equivalent PEG chains and fatty acids, but differentlinkages (shown in Table 5) were tested for stability at low pH, in therange of 1.4 to 2.1. Table 5 includes alternate names for each of thethree DAG-PEGs tested.

TABLE 5 DAG-PEGs with different linkages Linker Names Ac₂ PEG (n =12)-3-acetyl-1,2-rac-dioleoylglucerol PEG-12-Ac₂-GDO (glycerol dioleate)N₃ PEG (n = 12)-3-acetamido-dioleoylglycerol PEG-12-N₃-GDO (glyceroldioleate) S₂ PEG (n = 12)-3-N-(mercaptomethyl)-Propionamido-1,2-racdioleoylglucerol PEG-12-S₂-GDO (glycerol dioleate)

The selected DAG-PEGs were mixed with 10 mM phosphate buffer (pH 2.1) atfinal concentrations approximately 0.05 g/mL and incubated at 37° . Thesample solutions were tested on a GPC column with RI detector and thebreakdown fragments were further confirmed by LC-MS/MS. The stabilityprofiles are presented in FIG. 1.

In FIG. 1, (1) is PEG (n=12)-3-acetyl-1,2-rac-dioleoylglucerol, (2) isPEG (n=12)-3-acetamido-dioleoylglycerol and (3) is PEG(n=12)-3-N-(mercaptomethyl)-Propionamido-1,2-rac-dioleoylglucerol. (1)was stable, (2) was relatively stable, and (3) degraded quickly at a lowpH (in the range of human gastric pH, i.e., 1.4 to 2.1).

Example 25 Biocompatibility Experiments on DAG-PEGs

The use of adequate target cells for cytotoxicity testing of biomedicalmaterials has often been experimentally assessed with respect to theclinical relevance of the test results. An evaluation of new syntheticPEG-lipids with regard to biocompatibility is critically important forjudging the potential of new materials in biological-relatedapplications. Thus, the cytotoxicity of the novel PEG-lipids wasevaluated qualitatively by minimal essential medium testing with L929cell line. This was accomplished by first introducing the lipids withfresh 10% bovine serum (pH 7.2) to give a final concentration of 5mg/mL. The samples were then sterilized and extensively washed threetimes with sterile sodium chloride-phosphate buffer (pH 7.4) prior totransfer to individual 24-well tissue culture plates. Aliquots (1 mL) ofmouse fibroblasts (L929) suspension with 1.5×10⁴ cell/mL were seeded onthe sample membranes. After 48 h of culture, cellular constructs wereharvested, rinsed twice with sodium chloride-phosphate buffer to removefree cells, and adherent cells were fixed with 3.0% glutaraldehyde at 4°C. for 5 h. The samples were dehydrated through repeated rinsing withalcohol solutions and air-dried overnight. Dry samples were coated withgold for observation of cell morphology on the surface of the scaffoldsby SEM. No qualitative change in monolayer confluence and morphology wasobserved in the polyester samples relative to the positive control,indicating negligible cytotoxic response of the cells to the PEG-graftedlipids. Similar results were obtained for all PEG-lipids tested.

In addition, hemolysis of human red blood cells was performed to providea more quantitative cytotoxicity evaluation of the PEG-grafted lipids.Cell lysis caused by cytotoxic material leads to release of heme intosolution, which was detected by absorbance at 413 nm and compared tocontrol experiments performed in the absence of the synthetic material.Results in Table 6 for equivalent concentrations (20 mmol) of sodiumlauryl sulfate (SLS) and mPEG-600 were shown for the comparison. Lessthan 1.6% lysis was observed for all PEG-grafted lipids, with the lowestvalue (0.3%) measured for PEG 12-N₃-GDO. These results weresignificantly lower than PEG 600 monomethyl ether, the starting material(3.5%) and dramatically lower than those obtained for the surfactant SLS(80%), used as a comparative control.

TABLE 6 Comparison of Hemolysis Linker Lipid % Hemolysis Control 1 mPEG600 3.5 Control 2 SLS 80 N₁ PEG-12-N₁-GDO 0.3 N₂ PEG-23-N₂-GDO 0.5 N₃PEG-18-N₃-GDO 0.3 N₄ PEG-23-N₄-GDO 0.7 S₁ PEG-8-S₁-GDO 1.5 S₂PEG-18-S₂-GDO 1.2 S₃ PEG-12-S₃-GDO 1.5 O PEG-12-oxy-GDO 1.6 Ac₁PEG-18-Ac₁-GDO 1.0 Ac₂ PEG-12-Ac₂-GDO 1.1 N₁ PEG-12-N₁-GDM 0.4 N₁PEG-12-N₁-GDLO 0.3 S₃ PEG-12-S₃-GDM 1.5 S₃ PEG-12-S₃-GDLO 1.5 Ac₂PEG-12-Ac₂-GDM 1.0 Ac₂ PEG-12-Ac₂-GDLO 0.9 N₁ PEG-23-N₁-GDL 0.3 N₁PEG-12-N₁-GDP 0.3 Ac₂ PEG-23-Ac₂-GDL 0.8 Ac₂ PEG-12-Ac₂-GDP 0.9

Example 26 Rifampicine IV Injectable Solution

DAG-PEG lipid was added to a vessel equipped with a mixer propeller. Thedrug substance was added with constant mixing. Mixing continued untilthe drug was visually dispersed in the lipids. Pre-dissolved excipientswere slowly added to the vessel with adequate mixing. Mixing continueduntil fully a homogenous solution was achieved. A sample formulation isdescribed in Table 7, the targeted pH range was between 6.5 and 8.

TABLE 7 Ingredient mg/mL Rifampicine   4.0 PEG Lipid 40 Sodium HydroxideSee Below Lactic Acid 50 Purified Water qs 1 mL

The lipid may be PEG 12-GDO (oxyl linkage), PEG-12-acetamido (N₂)-GDO,PEG 12-GDM (oxyl linkage), PEG-12-acetamido (N₂)-GDM, PEG 12-N₃-GDLO,PEG 12-GDLO (oxyl linkage) or any combination thereof Sodium hydroxide(NaOH) is used to prepare a 10% w/w solution in purified water, and NaOHis used to adjust pH if necessary. The targeted pH is in a range of6.5.0 to 8.0.

Example 27 Bioavailability of Rifampicine Formulations

Groups of three male mice (B6D2F1) were used for the studies.Pharmacokinetics (PK) were performed on heparinized mouse plasma samplesobtained typically at 0 hr, 0.08 hr, 0.25 hr, 0.5 hr, 1 hr, 2 hr, 4 hr,8 hr, 16 hr and 24 hr after the bolus IV injection. Samples wereanalyzed using a HPLC-MS method. To determine the level of each drug,the drug was first isolated from plasma with a sample pre-treatment.Acetonitrile were used to remove proteins in samples. An isocraticHPLC-MS method was then used to separate the drugs from any potentialinterference. Drug levels were measured by MS detection with a multiplereaction monitoring (MRM) mode. PK data was analyzed using the WinNonlinprogram (ver. 5.2, Pharsight) compartmental models of analysis.

FIG. 2 shows comparison between mouse PK profiles of various Rifampicineformulations administered intravenously. DAG-PEG formulations of (1)PEG-12-acetyl-GDO and (2) PEG-12-acetamido-GDO of rifampicin, as well as(3) a rifampicin solution containing 5% dimethyl sulfoxide and 10%Cremophor EL were tested. The drug was administered intravenously andthe dosing strength was 20 mg/kg. The AUC (area under the curve) of theDAG-PEG formulations were (1) 878.6 μg·hr/mL and (2) 1061.5 μg·hr/mLversus 341.2 μg·hr/mL for the Cremophor EL solution (3).

FIG. 3 shows a comparison between mouse PK profiles from oraladministrations of the drug. The formulations and dose strength were thesame as in FIG. 2. The relative bioavailability (based onAUC_(0 to 24 hr) of PEG-12-acetyl-GDO) of the DAG-PEG formulations were(1) 71.6% and (2) 68.6% versus 29.8% for the Cremophor EL solution (3).

FIG. 4 shows a comparison among the drug level in lung after IVadministration with (1) PEG-12-acetyl-GDO and (2) PEG-12-acetamido-GDOrifampicin liposomes and (3) a rifampicin solution containing 5%dimethyl sulfoxide and 10% Cremophor EL. The dosing strength was thesame as in FIG. 2. After drawing blood for PK studies, mice weresacrificed at each time point and organs were removed. The drug was thenextracted with organic solvent and tested with the same procedure as forPK plasma. Lung is one of the targets for TB treatment. Significantamounts of the drugs were delivered to the lung by the DAG-PEGformulations. The accumulated amounts (μg·hr/mg) of rifampicin for theDAG-PEG formulations were (1) 8095.8 and (2) 8624.7 versus 3902.1 forthe Cremophor EL solution (3). The challenge is that the current solecommercial IV formulation requires administration of the drug within afew hours, otherwise the drug will be precipitated. Also, the drug isvery toxic. The tested liposomal formulations are stable suspensionswhich can keep at room temperature for months without precipitation andalso provide a better PK and lung delivery profile.

FIG. 5 shows a comparison among the drug level in liver after IVadministration from (1) PEG-12-acetyl-GDO, (2) PEG-12-acetamido-GDO ofrifampicin and (3) a rifampicin solution containing 5% dimethylsulfoxide and 10% Cremophor EL. The dosing strength was the same as inFIGS. 2 and 4. The accumulated amounts (μg·hr/mg) of Rifampicin curve)of the DAG-PEG formulations were (1) 34595.7 and 36889.9 (2) versus36177.2 for the Cremophor EL solution (3). The drug was not retained inthe liver while large amounts of the drugs were delivered to the lung bythe DAG-PEG formulations of (1) and (2).

The pharmacological activity of rifampin was considerably increased whenit was encapsulated in the liposomes. Rifampin-liposomes delivered twiceamounts into lung while having a similar profile in liver as compared toCremophor solution. The new liposome formulations can appreciablyincrease the therapeutic efficacy of rifampin. These results clearlydemonstrated that liposome targeting to macrophages can considerablyincrease the antitubercular activity of rifampin.

Example 28 Antifungal-Lipid Formulations

Tetrahydrofuran antibiotics are widely used as antifungal agents. Theyinclude the drug ketoconazole and are described in U.S. Pat. No.5,039,676. Newer tetrahydrofurans have been developed that are moreeffective and less toxic than ketoconazole. They are described in U.S.Pat. No. 5,661,151. The newer tetrahydrofurans include posaconazole,voriconazole and itraconazole.

In addition to the agents described in U.S. Pat. No. 5,661,151, theclass of tetrahydrofuran drugs includes a new tetrahydrofuran drug thatis referred to herein as equaconazole. Equaconazole has the followingstructure(s).

In this example, fungicide was combined in an aqueous solution withDAG-PEG lipids. A variety of drug-to-lipid ratios were tested, as wellas a variety of formation temperatures. After mixing, formulations wereallowed to stand at room temperature. The results are shown in Table 8.

TABLE 8 (a) Lipid w/Posaconazole Lipid Conc Drug Solubility (30 mgdrug/mL) (mg/mL) 25° C. 35° C. 45° C. 60° C. PEG-12-N₃-GDO 120 + ++ ++++ PEG-12-N₃-GDM 120 + ++ ++ +++ PEG-12-N₃-GDLO 150 + ++ ++ +++PEG-12-N₃-GDP 150 + ++ ++ +++ PEG-12-Ac₂-GDO 120 + ++ ++ +++PEG-12-Ac₂-GDM 120 + ++ ++ +++ Drug Solubilized @ 40° C. (b) Lipidw/Posaconazole L/D L/D L/D L/D L/D (30 mg drug/mL) Ratio = 1 Ratio = 3ratio = 5 ratio = 10 ratio = 20 PEG-12-N₃-GDO −/+ + ++ +++ ++PEG-12-N₃-GDM −/+ + ++ +++ ++ PEG-12-N₃-GDLO −/+ + ++ +++ +++PEG-12-N₃-GDP −/+ + ++ +++ +++ PEG-12-Ac₂-GDO −/+ + ++ ++ ++PEG-12-Ac₂-GDM −/+ + ++ ++ ++

Example 29 Equaconazole-Lipid Formulations

Equaconazole was combined in an aqueous solution with DAG-PEG lipids. Avariety of drug-to-lipid ratios were tested, as well as a variety offormation temperatures. After mixing, formulations were allowed to standat room temperature. The results are shown in Table 9.

TABLE 9 (a) Lipid w/Equaconazole Lipid Conc Drug Solubility¹ (30 mgdrug/mL) (mg/mL) 25° C. 35° C. 45° C. 60° C. PEG-12-N₃-GDO 120 + ++ ++++ PEG-12-N₃-GDM 120 + ++ ++ +++ PEG-12-N₃-GDLO 120 + ++ ++ +++PEG-12-N₃-GDP 150 + ++ ++ +++ PEG-12-Ac₂-GDO 120 + ++ ++ +++PEG-12-Ac₂-GDM 120 + ++ ++ +++ Drug Solubilized @ 40° C. (b) Lipidw/Equaconazole L/D L/D L/D L/D L/D (30 mg drug/mL) ratio = 1 ratio = 3ratio = 5 ratio = 10 ratio = 20 PEG-12-N₃-GDO −/+ + ++ +++ ++PEG-12-N₃-GDM −/+ + ++ +++ ++ PEG-12-N₃-GDLO −/+ + ++ +++ +++PEG-12-N₃-GDP −/+ + ++ +++ +++ PEG-12-Ac₂-GDO −/+ + ++ ++ ++PEG-12-Ac₂-GDM −/+ + ++ ++ ++

In Tables 8 and 9, “−” means not soluble; “−/+” means partially soluble;“+” soluble, “++” very soluble, “+++” most soluble. L/D ratio meanslipid to drug ratio.

Example 30 Antifungal Oral Solution

PEG lipid was added to a vessel equipped with a mixer propeller. Thedrug substance was added with constant mixing. Mixing continued untilthe drug was visually dispersed in the lipids. Pre-dissolved excipientswere slowly added to the vessel with adequate mixing. Mixing continueduntil fully a homogenous solution was achieved. A sample formulation isdescribed in Table 10.

TABLE 10 Ingredient mg/mL Antifungal Active 30.0 PEG Lipid 100 LacticAcid 50 Sodium Hydroxide See below Hydrochloric Acid See below SodiumBenzoate 2.0 Artificial Flavor 5.0 Purified Water qs 1 mL

The drug may be itraconazole, posaconazole, voriconazole orequaconazole. The lipid may be PEG-12-N₃-GDO, PEG-12-N₃-GDM,PEG-12-N₃-GDLO, PEG-12-N₃-GDP, PEG-12-Ac₂-GDO, PEG-12-Ac₂-GDM or anycombination thereof Sodium hydroxide is used to prepare a 10% w/wsolution in purified water. The targeted pH is in a range of 4.0 to 7.0.NaOH is used to adjust pH if necessary.

Example 31 Antifungal IV Injectable Solution

The IV solution was prepared as in Example 26, except that the targetedpH range was between 6.5 and 7.5. A sample formulation is described inTable 11.

TABLE 11 Ingredient mg/mL Antifungal Active  30.0 PEG Lipid 100  SodiumHydroxide See Below Lactic Acid 50 Purified Water qs 1 mL

The drug may be itraconazole, voriconazole, posaconazole orequaconazole. The lipid may be PEG-12-N₃-GDO, PEG-12-N₃-GDM,PEG-12-N₃-GDLO, PEG-12-N₃-GDP, PEG-12-Ac₂-GDO, PEG-12-Ac₂-GDM or anycombination thereof. Sodium hydroxide is used to prepare a 10% w/wsolution in purified water. The targeted pH is in a range of 6.5 to 7.0.NaOH is used to adjust pH if necessary.

Example 32 Pharmacokinetic Profile and Bioavailability of PosaconazoleFormulations

Groups of three male mice (B6D2F1) were used for the studies.Pharmacokinetics (PK) were performed on heparinized mouse plasma samplesobtained typically at 0 hr, 0.08 hr, 0.25 hr, 0.5 hr, 1 hr, 2 hr, 4 hr,8 hr, 16 hr and 24 hr after the bolus IV injection or oral feeding forposaconazole and at 0 hr, 0.08 hr, 0.25 hr, 0.5 hr, 1 hr, 2 hr, 4 hr, 8hr, 16 hr and 24 hr for itraconazole. Samples were analyzed using aHPLC-MS method. To determine the level of each drug, the drug was firstisolated from plasma with a sample pre-treatment. Acetonitrile were usedto remove proteins in samples. An isocratic HPLC-MS method was then usedto separate the drugs from any potential interference. Drug levels weremeasured by MS detection with a multiple reaction monitoring (MRM) mode.PK data was analyzed using the WinNonlin program (ver. 5.2, Pharsight)compartmental models of analysis.

FIG. 6 shows mouse PK profiles of posaconazole formulations with (1)PEG-12-acetamido (N₃)-GDO (1:3 drug to lipid ratio) and (2)PEG-12-acetamido (N₃)-GDM (1:5 drug to lipid ratio), (3)palmitoyl-oleayl phosphatidylcholine, or POPC, (1:1 drug to lipid ratio)and (4) a posaconale solution containing 5% dimethyl sulfoxide and 10%Cremophor. The drug was administered intravenously and the dosingstrength was 10 mg/kg. The AUC were 289.3 μg·hr/mL and 287.5 μg·hr/mLfor the DAG-PEG formulations (1) and (2), respectively, and 164.1μg·hr/mL and 193.2 μg·hr/mL for formulations of (3) and (4),respectively.

FIG. 7 shows mouse PK profiles of posaconazole formulations with (1)PEG-12-acetamido (N₃)-GDO (1:3, drug to lipid ratio) and (2)PEG-12-acetamido (N₃)-GDM (1:5, drug to lipid ratio), (3) a commercialproduct and (4) a posaconazole solution containing 5% dimethyl sulfoxideand 10% Cremophor. The drug was administered orally and the dosingstrength was 50 mg/kg. The relative bioavailability (based on theAUC_(0-24 hr) of the Cremophor formulation) were 53.8.% and 49.7% forthe formulations of PEG-DAG (1) and (2), 33.2% and 38.8% for theformulations of (3) and (4), respectively.

Example 33 Antifungal Topical Cream

PEG lipid was added to a stainless steel vessel equipped with propellertype mixing blades. The drug substance was added with constant mixing.Mixing continued until the drug was visually dispersed in the lipids ata temperature to 60°-65° C. Organic acid, Cholesterol and glycerin wereadded with mixing. Ethanol and ethyoxydiglycol were added with mixing.Finally Carbopol ETD 2020, purified water and triethylamine were addedwith mixing. Mixing continued until fully a homogenous cream wasachieved. The formulation is described in Table 12.

TABLE 12 Ingredient % Antifungal Active 1.0 PEG Lipid 5.0 Carbopol ETD2020 0.5 Ethyoxydiglycol 1.0 Ethanol 5.0 Glycerin 1.0 Cholesterol 0.4Triethylamine 0.20 Organic acid 10 Sodium hydroxide See below Purifiedwater qs 100

The drug may be itraconazole, posaconazole, voriconazole orequaconazole. The lipid may be PEG-12-N₃-GDO, PEG-12-N₃-GDM,PEG-12-N₃-GDLO, PEG-12-N₃-GDP, PEG-12-Ac₂-GDO, PEG-12-Ac₂-GDM or anycombination thereof. Organic acid may be lactic acid or pyruvic acid orglycolic acid. Sodium hydroxide is used to adjust pH if necessary. Thetargeted pH range was between 3.5 and 7.0.

Example 34 Antifungal Topical Solution

The topical solution was prepared as in Example 33, except that activewas first dissolved in organic acid and ethanol. A sample formulation isdescribed in Table 13.

TABLE 13 Ingredient % Antifungal Active 1.0 PEG Lipid 5.0 α-Tocopherol0.5 Organic acid 10.0 Ethanol 5.0 Sodium Benzoate 0.2 Sodium HydroxideSee Below Purified Water qs 100

The drug may be itraconazole, posaconazole, voriconazole orequaconazole. The lipid may be PEG-12-N₃-GDO, PEG-12-N₃-GDM,PEG-12-N₃-GDLO, PEG-12-N₃-GDP, PEG-12-Ac₂-GDO, PEG-12-Ac₂-GDM or anycombination thereof. Organic acid may be lactic acid or pyruvic acid orglycolic acid. Sodium hydroxide is used to adjust pH if necessary. Thetargeted pH range was between 3.5 and 7.0.

In another aspect, the invention comprises a method of solubilizing awater-insoluble agent, i.e., a drug compound that, because of lowsolubility in water, typically requires formulation with apharmaceutically acceptable carrier for effective delivery to anintended site of action. Such delivery may be intravenous, oral,topical, subdermal, sublingual, or any other mode of drug delivery. Theinvention also includes compositions for such delivery. Both the methodsand the compositions related to delivery of water-insoluble agentsemploy the PEG-lipid conjugates of the present invention and the methodsand materials described above.

While preferred embodiments of the present invention have beendescribed, those skilled in the art will recognize that other andfurther changes and modifications can be made without departing from thespirit of the invention, and all such changes and modifications shouldbe understood to fall within the scope of the invention.

1. A compound represented by the formula

where R1 is either —OH or —OCH3; where R2 and R3 are alkyl groups havingbetween 6 and 22 carbons; and where X is amino.
 2. The compound of claim1 where R2 and R3 are selected from the group consisting of oleate,myristate, linoleate and palmitate.
 3. The compound of claim 1 where thePEG chain consists of between about 6 and 45 subunits.
 4. The compoundof claim 3 where the PEG chain consists of between about 8 and 23subunits.
 5. The compound of claim 3 where the PEG chain consists ofbetween about 12 and 23 subunits.